# Gut bacterial metabolism of the side-chain of corticosteroids

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2024 · $184,675

## Abstract

Project Summary
The Sterolbiome Laboratory focuses on working out host-associated bacterial steroidogenic pathways. The focus
of the parent R01 is in identifying bacterial 17α-HSDH and 17β-HSDH involved in conversion of androstenedione
to the stereoisomers epitestosterone and testosterone, respectively. So far, we have discovered a 17α-HSDH
from the gut bacterium, Clostridium scindens, and 17β-HSDH from urinary isolates of Propionimicrobium
lymphophilum. These initial discoveries led to a manuscript that is currently under review in the journal Nature.
However, we are now focusing on identifying additional isoforms of bacterial 17α-HSDH and 17β-HSDH in
human stool samples (>200 samples). To accomplish this, we will use sequence agnostic and culture
independent functional metagenomic library screening. In this approach, community DNA is partially digested
and ligated into an expression vector before transformation into E. coli. Colonies are transferred to wells of
microplates containing bacterial medium and steroid substrates. After extraction of steroid products, hundreds
to thousands of wells need to be analyzed by liquid chromatography mass spectrometry. The Sterolbiome lab at
Illinois has a much larger collection of steroids than the Metabolomics Core on campus, and even core labs at
other institutions that specialize in steroid quantification. Using core labs is both time consuming, and results in
inconsistencies as different facilities utilize different instruments, columns, and solvent systems. The throughput
we are approaching with functional metagenomic subaims makes a dedicated instrument a necessity. Since
functional metagenomics is expected to result in identification of novel enzymatic reactions, we require MS/MS
application to produce unique daughter ions which act as “fingerprints” to assist in identifying unknowns. Knowing
the mass(es) of each peak is critical to differentiating steroid ions from other compounds and provides immediate
information on likely products. Without MS/MS, we would still need to submit most samples to core labs which
is the problem to begin with. Instead, we can develop a custom standardized workflow with the ThermoFisher
Vanquish Flex UHPLC coupled with VWD & IR, and TSQ Fortis Plus electrospray ionization (ESI) Triple
Quadrupole MS. PI Ridlon has support from both his Department and College and with matching funds, the
overall cost to NIGMS for the equipment has been significantly reduced.

## Key facts

- **NIH application ID:** 11100689
- **Project number:** 3R01GM145920-03S1
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **Principal Investigator:** Jason Michael Ridlon
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $184,675
- **Award type:** 3
- **Project period:** 2022-09-15 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11100689

## Citation

> US National Institutes of Health, RePORTER application 11100689, Gut bacterial metabolism of the side-chain of corticosteroids (3R01GM145920-03S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/11100689. Licensed CC0.

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